THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol 234, No. 7,July 1959

Printed in U.S.A.

The Hydrolysis of Carbobenzoxy-L-tyrosine p-Nitrophenyl

Ester by Various Enzymes*

CHARLES J. MARTIN, JULIUS GOLUBOW, AND A. E. AXELROD

WITH THE TECHNICAL ASSISTANCE OF ALBERT R. FRAZIER

From the Biochemistry Department, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania

(Received for publication, February 2, 1959)

cY-Chymotrypsin rapidly catalyzes the release of p-nitrophenol from carbobenzoxy-n-tyrosine p-nitrophenyl ester and the re- action has been made the basis of a spectrophotometric assay requiring only millimicrogram quantities of the crystalline en- zyme (1). Other nitrophenyl esters, e.g. of acetic acid (2), hy- drocinnamic acid (3), and carbobenzoxyglycine (4) are also hy- drolyzed by chymotrypsin despite the absence of structural parameters previously considered requisite in substrates sensitive to thii enzyme (5). As a generalization, an increase in the rate of the deacylation reaction is observed as the acyl contributor to the sensitive bond approaches the structure of an aromatic amino acid residue (3, 6). Trypsin has also been reported to hydrolyze p-nitrophenyl acetate (6), a compound of great struc- tural variance to other trypsin-sensitive synthetic substrates (5). The hydrolysis of this substrate by both chymotrypsin and tryp- sin represents another instance of the cross-reactivity of these two enzymes to the same or closely related substrates (T-11).

Other enzymes such as erythrocyte cholinesterase (12), eel cholinesterase (13), and the A-, B-, and C-esterases (14-16) are also capable of hydrolyzing p-nitrophenyl acetate and related esters, e.g. phenyl acetate. Since we are interested in the pos- sible use of acylated aromatic amino acid esters containing an aromatic alcohol as contributor to the sensitive bond for the assay of proteolytic enzyme activity in very small tissue samples, it was considered appropriate to determine if enzymes other than chymotrypsin could hydrolyze carbobenzoxy-n-tyrosine p-nitro- phenyl ester.

Preliminary reports of some of this material have appeared (17, 18).

EXPERIMENTAL

Materials-The CTNr preparation has been described else- where (1) and was dissolved in acetone before use.2 Other sub-

* This investigation was supported, in part, by Research Grants A-727 and A-2996 from the National Institutes of Health, United States Public Health Service, and by the Office of Naval Research under contract 1833(00), NR 101-412.

1 The abbreviations used are: BAL, 2,3-dimercaptopropanol (British anti-Lewisite) ; CTN, N-carbobenzoxy-n-tyrosine p-nitro- phenyl ester; DFP, diisopropylphosphofluoridate; TAME, Na- tosyl-n-arginine methyl ester; Tris, tris(hydroxymethyl)amino- methane.

2 An improved method for the synthesis of CTN is as follows. Equimolar quantities (1.0 mmole) of carbobenzoxy-n-tyrosine, p-nitrophenol, and N,N’-dicyclohexylcarbodiimide (Mann Re- search Laboratories, New York 6, New York) were added, in the

strates were commercial preparations. Trypsin (Lot No. T582, once crystallized), papain (Lot No. 5436, crystalline), carboxy- peptidase (Lot No. 597, 3 times crystallized), pepsin (Lot No. 623, twice crystallized), and wheat germ lipase (Lot No. 5519) were obtained from the Worthington Biochemical Corporation. Thrombin (bovine, topical) was obtained from Parke, Davis and Company. The potency of the enzyme, in terms of the NIH thrombin unit (T.U.) (19) was accepted as stated on the manu- facturer’s label (however, cf. (20)). Practically all of the experi- ments with thrombin were done with a single sample which con- tained 15.6 pg. of protein per T.U. A plasminogen preparation equivalent to the solution B of Kline (21) was prepared from human plasma Fraction III. A human plasmin preparation (Actase, Ortho Pharmaceutical Company) was also used. Fi- brinogen was obtained from the Warner-Chilcott Laboratories. Electric eel cholinesterase was obtained from Professor I. B. Wilson who stated that the total sample was capable of hydro- lyzing 350 pmoles of acetylcholine per minute. The preparation was dissolved in 1.0 ml. of 0.1 M KaCl and thii solution will be referred to as the “stock enzyme.” Insufficient material was available for a protein determination. Other enzyme prepara- tions were made up as follows: trypsin in 0.12 M CaC12, pepsin in 0.001 N HCI, carboxypeptidase in 10 per cent LiCl, plasmin in 0.15 M NaCl, and thrombin, papain, and wheat germ lipase in water.

Methods-The velocity of CTN hydrolysis was determined by measurement of the rate of appearance of p-nitrophenol (as the p-nitrophenolate ion) at 400 mp. Details of the assay procedure were as previously described (1) and reaction solutions contained 0.50 ml. of 0.20 M Tris buffer (pH 8.0), 1.00 ml. of 0.30 M cacl2,

0.35 ml. of methanol, 0.20 ml. of enzyme, 0.10 ml. of CTN (in acetone), and water to 3.00 ml. volume. The temperature was 30.0”. In the papain assays, CaClz was replaced with KC1 at a concentration of 0.30 M. BAL was used at a concentration of approximately 1O-5 M for the activation of papain and was added to the reaction solution immediately after papain addition and just before the addition of substrate. In experiments with other

order listed, to 10 ml. of tetrahydrofuran. After 3 hours at room temnerature. the nrecinitate that had formed was filtered off and the hltrate taken io dryness by vacuum distillation. Solution of the residue in ethyl acetate followed by its removal, in a vacuum, gave a crystalline product, which, after recrystallization from hot chloroform, melted at 157-158’; yield, 65 per cent.

The optical rotation in acetone ([a]: -16.3; 1.0 per, cent) was identical to that obtained with CTN prepared by a mixed an- hydride procedure (1).

1718

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

July 1959 C. J. Martin, J. Golubow, and A. E. Axehod 1719

enzymes, methanol and salt were omitted. The initial substrate concentration, ao, was between 4.0 and 4.5 X 1O-5 M when meth- anol was present in the reaction solution; in the absence of meth- anol, a0 was about 2.5 X 1O-6 M. Within the limit concentra- tions of methanol and salt employed, the value of ez& for the p-nitrophenolate ion was not affected. The CTN spontaneous hydrolysis rate, however, was increased approximately 1.5-fold in the presence of 11.7 volumes per cent methanol and 0.30 M KCl.

The hydrolysis of TAME, triacetin, and of acetylcholine was determined by potentiometric null point titration at 30” and pertinent details will be given in the text. The clotting of fibrin- ogen was measured at pH 7.0 (Tris buffer) and ionic strength 0.15 at 37” in solutions containing 66 fig. of clottablc protein per ml.

Trypsin, carboxypeptidase, and soybean inhibitor concentra- tions were determined by absorbance measurements at 280 rnp with the following extinction coefficients for a 1.0 per cent solu- tion: 14.4 for trypsin (22), 23 for carboxypeptidase (23), and 8.5 for soybean inhibitor (24). In other cases, protein concentration was determined by the method of Lowry et al. (25).

Initial velocities, ~0, defined as the increase in absorbancy at 400 rnp per second (where CTN was the substrate) or as the pmoles of substrate hydrolyzed per minute, were corrected for nonenzymic contributions to the total rate and were determined in the range wherein proportionality to enzyme concentration obtained.

Kinetic Studies: Trypsin, Papain, and Thrombin-Bate curves for the hydrolysis of CTN by trypsin, papain, and thrombin are given in Fig. 1. Trypsin hydrolyzed CTN according to first order kinetics; a plot of log so/a versus t giving a straight line (Fig. 1, inset graph). At a trypsin concentration of 40.2 mpg. per ml. and with a0 equal to 4.37 X 1O-6 M, the apparent first order reaction constant, k’, was calculated from the slope of the line to be 5.5 X 1O-3 sec.-l. In the nonenzymic hydrolysis of CTN, the first order reaction constant equals 6.4 X 1O-4 sec.-l.

The hydrolysis of CTN by thrombin did not fit first order re- action kinetics (Fig. 1). I f the reaction can be described by the integrated form of the Michaelis-Menten equation (26)

kret = 2.3 K, log so/a + (a0 - a) (1)

u-here ka is the velocity constant for the decomposition of the enzyme-substrate complex in the usual reaction formulation

h EfS:

J23 ES-E+P (2) k?

a plot of (a0 - a)/t versus log (ao/a)/t will give a straight line with a slope equal to -2.3 K,. When the data were plotted in this manner K, was calculated from the slope of the line to be 5.7 X 1O-6 ll~. A value similar to this was obtained by the solu- tion of a series of simultaneous equations derived from cxpres- sions of k3 as a function of K. at varying times of the reaction (27). Substitution of the above K, value into Equation 1 gave a straight line with zero intercept when the quantity 2.3 K. log so/a + (a0 - a) was plotted against t (Fig. 1, inset graph).

The data for the papain experiment gave an apparent fit to Equation 1, with the K, value for the reaction as determined by conventional means (see below), but this has little significance due to the considerable contribution to the total rate of the BAL- induced liberation of p-nitrophenol from CTN.

Activity was proportional to the trypsin, papain, and thrombin concentration over a IO-fold range or greater (Fig. 2A).

Trypsin catalyzed the hydrolysis of CTN optimally at pH 7.8 (Fig. 2B). This agrees with the effect of pH on the hydrolysis of other substrates sensitive to this enzyme (28).

The pH activity curves for the hydrolysis of both TAME and CTN by papain are given in Fig. 2B. Above pH 7.0, the rate of TAME or CTN hydrolysis decreased. Kimmel and Smith (29) have reported a somewhat different pH curve for the papain- catalyzed hydrolysis of TAME; above or below pH 6, activity towards this substrate is considerably decreased.

The maximal velocity of CTN hydrolysis by the thrombin preparation occurred at pH 8.5 (Fig. 2Q. The pH optimum for the hydrolysis of TAME by this enzyme in the presence of a Tris buffer has been reported to be at about 9 (30). When borate or phosphate buffers are used (30) or in the absence of buffer (20), the optimum is shifted to pH 8 or lower.

The effect of CTN concentration on the rate of hydrolysis by trypsin, papain, and thrombin was also studied. In these ex- periments, thrombin activity was measured in reaction solutions containing 11.7 volumes per cent methanol and 0.30 M KC1 to

0.2-

4 6 I 4 I ! I I I I 2 4 6 8 IO 12

Minutes

FIG. 1. Rate curves for the hydrolysis of CTN by trypsin (O-O), papain (A-A), and thrombin (0-O). In the thrombin-catalyzed reaction, a0 equaled 2.06 X lO+ M; otherwise, a0 was 4.37 X 10-S M. Enzyme concentrations: trypsin, 40.2 qg. per ml.; papain, 1.35 pg. per ml. ; thrombin, 0.66 T.U. per ml. In- sert graph: the fit of the rate curves from the trypsin- and throm- bin-catalyzed reactions to the first order and integrated Michaelis- Menten equation, respectively. For trypsin (O-O), F .= log a,/a (right ordinate); for thrombin (+--a), F = [2.3 K. log a0la + (a0 - a)] X lo5 (left ordinate). The time scale for the insert graph is numbered above the abscissa line. See text for other details.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

1720

I t I I 0 0.4 0.8

Enzyme Concentration 1.2

Hydrolysis of Carbobenxoxy-L-tyrosine p-Nitrophenyl Ester Vol. 234, No. 7

I I ’ I c

I 1 I I I L 6 7 8 9

PH

1 J

FIG. 2A. Velocity of CTN hydrolysis as a function of enzyme concentration. Abscissa value of unity equivalent to 74.4 mpg. of trypsin (O-O), 2.16 pg. of papain (A--A), and 1.0 T.U. (0-O) per ml.

B. Velocity of CTN hydrolysis as a function of pH; trypsin (O-O), papain (A-A), thrombin (0-O). The solid triangles (A-A) were derived from the papain-catalyzed hy- drolysis of TAME (a~, 0.025 M) in the presence of 6.6 X 1e3 M BAL.

o. x IO5 M

FIG. 3. The dependence of the velocity of CTN hydrolysis upon substrate concentration. Data Dlotted according to Woolf (31) : trypsin (O-O) and thrombih (O---O), lek ordinate ‘add lower abscissa; papain (A-A), right ordinate and upper ab- scissa.

permit of comparisons with the trypsin and papain data and to enable greater latitude in the range of a0 used. The data are plotted as ao/vo against a0 (31) (Fig. 3). From the slopes and intercepts of the subjectively drawn lines, values for K, and the specific rate constant, ks, were calculated, the latter on the basis of one active site per molecule. The data are given in Table I and are compared with previous results obtained with a-chymo- trypsin (1) and with corresponding constants for the hydrolysis of other substrates by these enzymes. A discussion of the data in Table I will be deferred until later in the text.

Trypsin Experiments-It has been reported that trypsin prep- arations are often contaminated with enzymic activity displaying the catalytic characteristics of chymotrypsin (35). On the basis of its activity towards N-acetyl-L-tyrosine ethyl ester at pH 8.0, and assuming that the contaminant was chymotrypsin, the trypsin preparation used in these experiments contained about 2.5 per cent by weight of this enzyme. However, on the basis of weight considerations alone, trypsin can be considered to be an effective catalyst of CTN hydrolysis, e.g. equal values of vo are obtained with either 10.0 mpg. of trypsin per ml. (cf. Fig. 2A) or with 2.3 mpg. of chymotrypsin per ml. (1).

Incubation of trypsin with either the lima bean inhibitor, the bovine plasma trypsin inhibitor, or ovomucoid completely in- hibited CTN hydrolysis. Determination of remaining enzyme activity in the presence of increasing concentrations of the soy- bean inhibitor gave essentially parallel inhibition curves with both TAME and CTN as substrates (Fig. 4). From the inter- cept value on the abscissa, 0.70 to 0.75 mg. of soybean inhibitor is required to inhibit 1 .O mg. of trypsin. Green (36) has reported a similar value (0.76 mg. of soybean inhibitor per mg. of trypsin) from data obtained with AT”-benzoyl-L-arginine ethyl ester as substrate.

Incubation of trypsin with DFP completely inhibited CTN hydrolysis.

Thrombin’ Experiments-Two different samples of thrombin did not differ in their ability to hydrolyze TAME and CTN (Table II). It has been reported that the soybean inhibitor, while effective against plasmin (37), does not produce inhibition of thrombin with TAME as substrate (30). This observation was confirmed and it was also demonstrated that the soybean inhibitor had no effect on CTN hydrolysis by the thrombin prep- aration (Table II). However, DFP will inhibit thrombin activ- ity (38, 39). Incubation of thrombin with DFP (0.01 M) at pH 8.0 for 5 minutes at 30’ before assay resulted in a loss of 96 and 88 per cent of the activity towards CTN and TAME, respec- tively. In the presence of 0.025 L{ TAME, CTN hydrolysis was completely abolished.

Ehrenpreis and Scheraga (20) have reported that Parke, Davis thrombin preparations contain a probable enzyme component, EI,, which catalyzes the disruption of the fibrin monomer formed as the result of thrombin action on fibrinogen. This enzyme was much more stable to low pH values than was thrombin; its ac- tivity being decreased about 2-fold upon exposure of a thrombin preparation to pH 3.48 for 10 minutes (temperature unspecified) as opposed to a greater than 99 per cent loss in thrombin activity (20). In a similar experiment at 30”, 95 per cent of the total thrombin activity was destroyed as determined with either CTN or TAME as test substrates (Table II). This would indicate that the postulated EI, (20) was not responsible for CTN hy- drolysis.

The effect of heating at 55” upon the enzyme activity of the thrombin preparation was followed by activity determinations against CTN, TAME, and fibrinogen (Fig. 5). The loss of ac- tivity towards CTN with increasing periods of heating paralleled the decreased activity towards TAME and fibrinogen.

When thrombin was chromatographed by the technique of Rasmussen (40)) the distribution of purified thrombin in various fractions determined with TAME as the test substrate coincided with the activity profile towards CTN.

Papain Experiments-Complete activation of papain can be obtained in the presence of a reducing agent, such as cysteine,

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

1721 July 1959 C. J. Martin, J. Golubow, and A. E. Axelrod

TABLE I Kinetic constants for hydrolysis of CTN and other substrates

All rate measurements with CTN as substrate were made at pH 8.0 and p/2, 0.3 in 11.7 volumes per cent methanol. ture was 30”. Molecular weights used were 24,000 for trypsin (32) and 20,500 for papain (33).

The tempera-

Cl-N TAME

(Y x”ioq ka

(M 2106) ka

@x.-q (sec.-*)

Trypsin. . 15 290 49oc 187C Thrombin...................... 8.6 22d 110’ 9.2df 0 Papain. 0.66 1.5 Chymotrypsin. 3.2 480

6 Na-benzoyl-n-argininamide at pH 5.2, 38”, 10 volumes per cent methanol (34). b N-acetyl-n-tyrosine ethyl ester at pH 8.0, 25”, 0.10 M CaC12, no methanol (1). c At pH 8.0, 25”, 0.10 M CaC12, no methanol. d In (moles 1.-l min.-l T.U.-1 ml.-‘) X 106. 8 At pH 8.0, 30”, no methanol. The ionic strength varied from 0.001 to 0.02.

Mg. Soybean Inhibitor per Mg. Trypsin

FIQ. 4. The inhibition of the trypsin-catalyzed hydrolysis of TAME and CTN by soybean inhibitor: (A-A), CTN as sub- strate; (O-O), TAME as substrate. In the TAME assays, a0 was about 0.01 M and CaClt was present at 0.10 M. The pH was 8.0.

and a divalent cationic chelating agent, such as ethylenediamine- tetraacetate (29) or in the presence of BAL alone (34). Alter- natively, passage of papain through a mixed bed ion exchange column (41) permits one to obtain fully activated papain free from reducing agents and heavy metal cations. The effect of BAL on the papain-catalyzed hydrolysis of CTN is shown in Fig. 6. In the absence of BAL, hydrolysis of CTN did not occur. Approximately 6.6 X 1O-5 M BAL was necessary for the maxi- mal activation of papain. As reported previously (l), BAL alone induces the release of p-nitrophenol from CTN (Fig. 6).

Iodoacetamide and p-chloromercuribenzoate are known to completely inhibit papain activity (29). Incubation of papain

-

--

_-

-

BAA” I ATEE”

(Id %O’)

5000

ka (sec.-l) (M x”iOS)

ka (sec.-l)

11.2 65 174

TABLE II Comparison of TAME and CTN hydrolysis by thrombin

Reaction solutions for TAME assays contained about 0.025 M

TAME, 0.001 M Tris buffer, and 8.3 T.U. per ml. CTN assays were conducted in similar reaction solutions but with the buffer con- centration increased (cf. “Experimental”) and with CTN equal to 2.3 X lO+ M. Enzyme was present at 0.32 T.U. per ml. The pH was 8.0 and the temperature was 30”. Thrombin plus soybean inhibitor (1.2 pg. per T.U.) or DFP (0.01 M) was incubated at 30” for 5 minutes before addition to the reaction solutions. Activity is expressed as the pmoles of substrate hydrolyzed per minute per T.U. Thrombin preparation A contained 15.6 pg. of protein per T.U.

Treatment CTN

[k&,. X 10’

None, preparation A. . None, preparation B Preparation A plus soybean in-

hibitor . Preparation A plus DFP.. Preparation A plus TAME (0.025

M)

Preparation B, exposed to pH 3.5 for 10 min. at 30”.

16.6 16.0

16.6 0.70

0.00

0.81

[kol;~ x 10~ . .

10.8 10.8

10.8 1.3

0.60

at pH 7.0 for 30 minutes at lo in the presence of either of these two compounds, followed by dilution and assay (final inhibitor concentration, 8 X 1O-6 M), demonstrated that activity toward TAME and CTN had been abolished. Although papain cata- lyzes the hydrolysis of an ester substrate, DFP will not inhibit this enzyme (29). Incubation at pH 8.0 for 3 hours at lo with 0.002 M DFP before assay produced no decrease in the rate of either TAME or CTN hydrolysis as compared to the rate ob- tained with papain similarly treated but with DFP absent.

In all of the papain assays, appropriate corrections for the con- tribution of the BAL-induced release of p-nitrophenol to the total rate were applied. A number of the above experiments were also done with the use of cysteine and ethylenediaminetetra- acetate as replacements for BAL. The results were comparable in each case.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

1722 Hydrolysis of Carbobenzoxy-L-tyrosine p-Nitrophenyl Ester Vol. 234, No. 7

A I I I I I I I I I

0 2 4 6 8 IO Minutes at !XP

FIQ. 5. The heat-inactivation of thrombin. Aliquots of throm- bin (250 T.U. per ml.), in about 15 ml. volume Pyrex glass-stop- pered tubes were immersed in a bath at 55” for times indicated, rapidly chilled, and diluted as necessary for assay against TAME (O-O), CTN (A-A), and fibrinogen (0-O). Condi- tions for TAME assay as in Table II. See “Experimental” for conditions of fibrinogen-clotting assays.

5*o*

0 4 8 12 [BALI x 10~ M

FIG. 6. The effect of BAL on the papain-catalyzed hydrolysis of CTN. Papain concentration, 3.6 pg. per ml.; ae, 4.58 X lO+ XC. The lower curve represents the effect of BAL on the hydrol- ysis of CTN in the absence of papain.

Experiments with Plasmin-Plasminogen, upon treatment with streptokinase or other activators (42, 43), yields plasmin, an enzyme capable of hydrolyzing TAME, L-lysine ethyl ester, and casein, and active in the dissolution of a fibrin clot. The plas-

.c 80-m

.I t a E

.? 60- ;: 2

Days FIG. 7. The autocatalytic conversion of plasminogen to plasmin.

Increase in plasmin activity followed with TAME (O-O), L-lysine ethyl ester (O-----O), and CTN (O-0) as substrates. Activation mixture contained 8.0 ml. of plasminogen (11.6 mg. protein per ml.), 0.42 ml. of 1.0 M phosphate buffer (pH 7.6), 8.42 ml. of glycerol, and 0.1 ml. of plasmin (Actase). The temperature was 37’. At zero and other indicated times, aliquots were re- moved and added to reaction flasks containing either 0.02 M TAME (pH 8.0), 0.02 M L-lysine ethyl ester (pH 6.5), or CTN (2.5 X 10-S M). With CTN as substrate, 0.2 ml. of activation mixture used per reaction; in the other assays, 0.4 ml. diquots were used per 3.0 ml. reaction volumes.

minogen to plasmin transformation can also be achieved by an autocatalytic reaction in the absence of activators (44).

Incubation of plasminogen with CTN did not result in the re- lease of p-nitrophenol. Hydrolysis did occur, however, if plas- minogen was first preincubated with streptokinase (800 units) at pH 8.0 for 5 minutes at 37” before addition to the CTN re- action solution. Streptokinase alone did not hydrolyze this sub- strate. Parallel experiments with TAME as the test substrate gave the same results.

During the autocatalytic activation of plasminogen, the rate of increase in activity toward TAME, L-lysine ethyl ester, and CTN approximately paralleled each other although a lag in the appearance of CTN hydrolyzing activity was noticeable (Fig. 7). When an aliquot was removed from the activation mixture at the end of 44 hours and dialyzed against 0.01 N HCI at 3 to 4’ for 18 hours, it was found that about 50 per cent of the activity had been lost as determined with either CTN or TAME as the test substrate. This loss of plasmin activity upon dialysis is at variance with reports by other investigators (44).

L-Lysine ethyl ester and TAME are effective inhibitors of casein digestion by plasmin (37). We have also found that these compounds inhibit the plasmin-catalyzed hydrolysis of CTN. At concentrations of 0.016 M TAME or L-lysine ethyl ester in the reaction solution, CTN hydrolysis was decreased 16 and 79 per cent, respectively.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

July 1959 6. J. Martin, J. Golubow, and A. E. Azelrod 1723

Plasmin can be inhibited by organophosphatcs (45). When plasmin was incubated with DFP under less than optimal con- ditions, the inhibition produced was found to be the same with either TAME or CTN as test substrates.

Experiments with Carboxypeptidase, Wheat Germ Lfpase, Pep- sin, and Eel Cholinesterase-Although carboxypeptidase-sensitive substrates possess a free carboxyl group, this enzyme will hy- drolyze the ester substrate, benzoyl-glycyl-/%phenyllactic acid (5). Incubation of carboxypeptidase (0.1 mg. per ml.) with CTN resulted in the rapid release of p-nitrophenol. However, the reaction was completely inhibited by DFP and was not af- fected by 1 , lo-phena.nthroline. DFP does not inhibit carboxy- peptidase (46) whereas 1 , 1 0-phenanthroline will inhibit the en- zyme (47) and thus, hydrolysis of CTN by the carboxypeptidase preparation was probably due to contaminating chymotrypsin and trypsin (35).

The ability of a wheat germ lipase preparation to hydrolyze CTN was studied in experiments with not only this substrate but also by parallel assays with triacetin (48) as substrate. The hydrolysis of triacetin was measured at pH 7.6 with substrate at about 0.05 M. Incubation of the lipase with iodoacetamide (0.01 BI) at pH 6.6 for 1 hour at 35’ produced comparable de- creases in the velocities of hydrolysis of both substrates. Incu- bation with p-chloromercuribenzoate (lo-’ M) resulted in a Z-fold greater inhibition of triacetin hydrolysis as compared to the in- hibition of CTN hydrolysis. However, Singer (49) has reported that the degree of wheat germ lipase inhibition by p-chloromer- curibenxoate varies with the substrate of assay. Exposure of the lipase preparation to 50” for 15 minutes caused a 50 per cent de- crease in the hydrolysis of triacetin. The rate of CTN hydrolysis was not affected. With more drastic conditions (10 minutes at 62’), the lipase preparation had lost over 90 per cent of its ac- tivity towards triacetin whereas the rate of CTN hydrolysis was decreased by only 10 per cent. Thus, it would appear that if the enzyme capable of triacetin hydrolysis can attack CTN, it does so at a very slow rate and that the wheat germ lipase prep- aration contains a,n additional enzyme capable of hydrolyzing CTN.

It has been previously reported that pepsin, at an approximate concentration of 33 pg. per ml., was unable to release p-nitro- phenol from CTN at pH 8.0 (1). This was one of a number of proteins tested at this pH to demonstrate that CTN hydrolysis by chymotrypsin was not due to a nonspecific acylation reaction. The ability of pepsin to hydrolyze CTN was reinvestigated in unbuffered reaction solutions containing 0.15 mg. of pepsin per ml. at pH 3.2 and 6.7. At these pH values, the change in ab- sorbancy at 275 nm was used to detect the disappearance of CTN (1). However, even at the lower pH, hydrolysis of CTN by pep- sin was not observed.

The activity of the cholinesterase preparation was measured at pH 8.0 with acetylcholine bromide (0.004 af) as substrate in reaction solutions containing 0.001 111 Tris buffer, 0.1 of NaCl, 0.04 RI MgC12, a,nd enzyme at a concentration of 2.5 X 1O-4 ml. stock enzyme (cj. lLExperimental”) per ml. (50). In our assays we found that 1.0 ml. of stock enzyme was capable of hydro- lyzing 325 pmoles of acetylcholine per minute, in good agreement with the stated potency (cf. “Experimental”). In the presence of acetone (3.3 volumes per cent) the activity was decreased to 185 pmoles per minute per ml.

The hydrolysis of CTN by the cholinestcrase preparation was studied under identical conditions of acetone, NaCI, and MgC12

concentration with substrate equal to 2.5 X low5 M but with buffer concentration increased (cj. “Experimental”) and with stock enzyme at 330 X 1O-4 ml. per ml. One ml. of the enzyme preparation was capable of hydrolyzing 6.8 X 10e3 pmole of CTN per minute. However, at the enzyme concentration used (132-fold greater than for acetylcholine hydrolysis), the velocity of the reaction was only l.Cfold greater than the nonenzymic rate. Despite the low order of activity observed, it was com- pletely abolished by preincubation of the stock enzyme solution at lo for 2 hours with DFP (1.6 x 1O-s nf).

DISCUSSION

Evidence has been presented which would indicate that tryp- sin, papain, thrombin, and plasmin catalyze the release of p-nitro- phenol from CTN. Carboxypeptidase, wheat germ lipase, cho- linesterase, and pepsin were ineffectual in catalyzing this reaction. The first group of enzymes thus share with cY-chymotrypsin (I), the ability to hydrolyze an acylated aromatic amino acid ester, despite the fact that the most sensitive substrates for these en- zymes contain a basic amino acid residue, e.g. TAh1E. The ap- parent breakdown of differentiation in substrate specificity pat- terns, although less marked for papain than for trypsin, thrombin, and plasmin since the former has been reported to hydrolyze N-acetyl-L-tyrosinamide (29), is not believed to represent the consequences of CTN acting as a nonselective acylating agent. I f this were true, one would expect that many other proteins could mediate the liberation of p-nitrophenol from CTN al- though, in this, and in a preceding paper (l), this was found not to occur. In every example with which we are concerned, the protein concentration necessary for the demonstration of CTN hydrolysis was lower than that necessary for the demonstration of hydrolysis of an acceptable test substrate. It should also be mentioned that incubation of chymotrypsin with acetyl-m- phenylalanine p-nitrophenyl ester resulted in the rapid release of p-nitrophenol in an amount corresponding to 50 per cent hy- drolysis.3 The remaining isomer, presumably the D-form, was

either not hydrolyzed or hydrolyzed at a very slow rate. Inspection of the data in Table I reveals that, in every case,

K, for CTN hydrolysis is lower than that of a comparison sub- strate of accepted sensitivity. In addition, values of the specific rate constant, ks, are higher than that observed with the com- parison enzyme-substrate system with the exception of the hy- drolysis of N”-benzoyl-L-argininamide by papain. In this case, ka is somewhat greater than that found for the papain&TN sys- tem although, as with other systems listed, reaction parameter differences do not permit of rigorous comnarison. Kimmel and Smith (29) have reported that papain hydrolyzes this amide suh- strate at an over-all velocity essentially equivalent to that ob- tained with TAME; a result markedly different from the much lower susceptibility of an amide substrate, as compared to the corresponding ester, when the enzyme is trypsin or chymotrypsin (5). In a recent paper by Smith and Parker (51) values for Ii’, of 0.04 1\1 and for k~ of 9 sec.-l (both values estimated from graph- ical plots) were reported for the papain-catalyzed hydrolysis of N”-benzoyl-n-arginine ethyl ester at pH 8.0 and 25”; values very similar to those reported for the hydrolysis of the amide analog (Table I). In view of the great dissimilarity in K, for the hy- drolysis of CTN and N”-benzoyl-L-argininamide by papain, and the approximately equal values for IT23, the data suggest support

3 Unpublished results.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

Hydrolysis of Carbobenxoxy-L-tyrosine p-Nitrophenyl Ester Vol. 234, No. 7

for the concept that both substrates funnel through the same intermediate complex, i.e. a thiol ester complex of enzyme plus substrate, as suggested by Stockell and Smith (34) and further elaborated by Smith (52). Gutfreund (53) has suggested a sim- ilar rate determining step during the hydrolysis of N”-benzoyl- n-argininamide by ficin.

As would be expected from the correspondence of CTN to the structural requirements for a chymotrypsin substrate (5), chymo- trypsin hydrolyzes this substrate at a rate greater than the other enzymes listed. The high k3 value for the trypsin-catalyzed hydrolysis of CTN, however, compares favorably with the chy- motrypsin-CTN system, 290 sec.-l versus 480 sec.-l. Certainly, steric factors alone cannot explain this result. For instance, Schwert and Eisenberg (54) found that in the trypsin-catalyzed hydrolysis of a series of N”-benzoyl-L-arginine esters, both K, and ka remained constant when R in RlCOOR was either a methyl, isopropyl, cyclohexyl, benzyl, or an a-glyceryl group. One possibility would be that the susceptibility of acylated non- basic amino acid esters to trypsin hydrolysis would be governed, in part, by the electron density of the ethereal oxygen atom. Such a reduction in the electron density would be expected to occur in an ester containing the p-nitrophenoxide group. The leveling effect of this group upon specificity to the amino acid residue is also indicated by the fact that trypsin, chymotrypsin, and papain will also hydrolyze the p-nitrophenyl esters of car- bobcnzoxyglycine and carbobenzoxy-L-methionine (18). Other examples are the hydrolysis of p-nitrophenyl acetate by chymo- trypsin (2) and trypsin (6), and the hydrolysis of the correspond- ing esters of isobutyric, trimethylacetic, hydrocinnamic, and hippuric acid by chymotrypsin (3). Acylation and subsequent deacylation of the reactive site in &chymotrypsin have also been observed with p-nitrophenyl acetate, aliphatic acid anhydrides, and aromatic acid chlorides (6). The acylated intermediate formed with the anhydrides would probably be structually sim- ilar to that formed during the hydrolysis of n-fatty acid esters of hydroxybenzoic acid (9). A decrease in the velocity of hy- drolysis occurs when an ethoxide group is substituted for the p-nitrophenoxide group in CTN. With chymotrypsin as the test enzyme, ka equals 13 sec.-l and K, has the value of 0.04 ~~

(compare with values for CTN, Table I). It is extremely doubt- ful if catalysis of carbobenzoxy-L-tyrosine ethyl ester could be detected w-ith even large quantities of trypsin.

Further discussion of these questions must be deferred until additional experimental evidence can be obtained concerning the significance of the alcohol contributor to the susceptible bond in an ester substrate in relation to the ease of hydrolysis.

In an essentially aqueous solution, the release of p-nitrophenol during the thrombin-catalyzed hydrolysis of CTN follows a time course predicted by the integrated Michaelis-Menten equation. Under such conditions, K. was determined graphically from a single rate curve (cf. “Results”) to be 0.57 X 10m6 M. From the plot of 2.3 K, log so/a + (a~ - a) versus 2 (cf. Fig. I), k~ can be calculated from the slope of the line and the enzyme concen- tration to be 2.4 x 1O-5 moles 1.~’ min.-l T.U.-1 ml.-‘. Compar- ison of these values with those listed in Table I (K,, 8.6 x 10-5; ka, 22 x lo-?, obtained from measurements of vo at ionic strength 0.3 in reaction solutions containing 11.7 volumes per cent meth- anol shows that under these later conditions both K, and ka are increased in magnitude. An increase in the over-all velocity of

4 Unpublished results.

p-nitrophenyl acetate hydrolysis by chymotrypsin has been re- ported to occur upon the addition of methanol (55).

The lack of reactivity of pepsin and carboxypeptidase towards CTN would agree with the present knowledge of their specificity requirements toward synthetic substrates (28). Attention might be called to the fact that, in these two cases, the test systems con- tained enzyme at concentrations of approximately 0.1 to 0.15 mg. of protein per ml., yet the liberation of p-nitrophenol was not observed.

Substrates for true cholinesterase contain the acetyl or pro- pionyl grouping but do not require choline as the alcohol con- tributor to the sensitive bond (56). For example, eel cholines- terase hydrolyzes p-nitrophenyl acetate at a rate approximately 0.1 that observed for acetylcholine (13). Since we observed that the cholinesterase preparation used in our studies hydrolyzed CTN at a rate approximately l/27,000 of that observed for acetyl choline, CTN cannot be considered to be a substrate for this enzyme.

In view of the fact that CTN is hydrolyzed by such diverse enzymes as chymotrypsin, trypsin, papain, thrombin, and plas- min, its use as a test substrate should be employed with caution. As indicated in this paper and elsewhere (l), it can be used as a substrate for enzyme activity determinations in those situations wherein either highly purified enzymes are employed or prelim- inary experimentation has shown that CTN (or another acylated amino acid p-nitrophenyl ester) will adequately serve as an in- dicator for the activity of an enzyme in a less purified state. Examples of the latter application are the assay of partially pu- rified thrombin and plasmin.

SUMMARY

The hydrolysis of i\T-carbobenzoxy-n-tyrosine p-nitrophenyl ester by a number of different enzyme preparations of varying specificity requirements has been investigated. The velocity of hydrolysis was determined by measurement of the rate of appear- ance of p-nitrophenol at 400 rnp. Trypsin, papain, thrombin, and plasmin catalyze the hydrolysis of this substrate despite the fact that substrates containing a basic amino acid residue, e.g. N*-tosyl-n-arginine methyl ester, are considered the most sensi- tive known substrates for this group of enzymes. The kinetics of N-carbobenzoxy-L-tyrosine p-nitrophenyl ester hydrolysis by trypsin, papain, and t,hrombin have also been investigated. Other enzymes studied, viz. carboxypeptidase, wheat germ li- pase, pepsin, and electric eel cholinesterase were not observed to hydrolyze N-carbobenzoxy-L-tyrosine p-nitrophenyl ester.

Acknowledgments-We wish to thank Drs. II. E. Carnes and J. K. Weston, Parke, Davis and Company, Det.roit, Michigan, for a generous supply of thrombin; Dr. B. E. Sanders, Merck Institute for Therapeutic Research, West Point, Pennsylvania, and Dr. T. D. Gerlough (by authorization of J. N. Ashworth, American National Red Cross), E. R. Squibb and Sons, New Brunswick, New Jersey, for samples of human plasma Fraction III; Dr. J. M. Ruegsegger, American Cyanamid Company, Led- erle Laboratories Division, Pearl River, New York, for Varidase, a source of streptokinase; Dr. Jessica Lewis for a commercial sample (Actase) of plasmin; Dr. H. Tauber, Venereal Disease Experimental Laboratory, United States Public Health Service, University of North Carolina, School of Public Health, Chapel Hill, North Carolina, for a sample of lima bean inhibitor; Dr.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

July 1959 C. J. Martin, J. Golubow, and A. E. Axelrod 1725

M. Laskowski, Department of Biochemistry, Marquette Univer- ment of Neurology, College of Physicians and Surgeons, Colum- sity, School of Medicine, Milwaukee, Wisconsin, for a sample of bia University, New York, New York, for a sample of electric bovine plasma trypsin inhibitor; and Dr. I. B. Wilson, Depart- eel cholinesterase.

REFERENCES

1.

2.

3.

4.

5. 6.

7.

8.

9. 10. 11.

12.

13.

14. 15. 16.

17.

18.

19.

20.

21. 22.

23.

24. 25.

26.

27. 28.

MARTIN, C. J., GOL~BOW, J., AND AXELROD, A. E., J. Biol. Chem., 234, 294 (1959).

HARTLEY, B. S., AND KILBY, B. A., Biochem. J., 50,672 (1952); 66, 288 (1954).

MCDONALD, C. E., AND BALLS, A. K., J. Biol. Chem., 227, 727 (1957).

DIXON, G. H., AND NEURATH, H., Federation Proc., 16, 173 (1957).

NEURATH, H., AND SCHWERT, G. W., Chem. Revs., 46,69 (1950). DIXON, G. H., AND NEURATH, H., J. Biol. Chem., 226, 1049

(1957). SCHWERT, G. W., NEURATH, H., KAUFXAN, S., AND SNOKE, J.

E., J. Biol. Chem., 172, 221 (1948). RAVIN. H. A.. BERNSTEIN, P., AND SELIGMAN, A. M., J. Biol.

Chewi, 208,‘l (1954). HOFSTEE, B. H. J., Biochim. et Biophys. Acta, 24, 211 (1957). DOHERTY, D. G., J. Am. Chem. Sot., 77, 4887 (1955). SHAPIRA, k., AN; DOHERTY, D. G., Federation Proc., 16, 352

(1956). MOUNTER, L. A., AND WHITTAKPR, V. P., Biochem. J., 64, 551

(1953). BERGMANN, F., RIMON, S., AND SEGAL, R., Biochem. J., 68,

493 (1958). ALDRI~GE, .W. N., Biochem. J., 63, 110 (1953). ALDRIDGE. W. N.. Biochem. J.. 63. 117 (1953). BERGMAN;, F., F&GAL, R., AND ‘RI&ON, S:, B&hem. J., 67, 481

(1957). MARTIN, C. J., GOLUBOW, J., AND AXELROD, A. E., Biochim.

et Biophys. Acta, 27, 430 (1958). GOLUBOW, J., AND MARTIN, C. J., Federation Proc., 17, 231

(1958). Minimum requirements for dried thrombin, National Institutes

of Health, Division of Biologics Control, Bethesda, Mary- land, 1946.

EHRENPREIS, S., AND SCIIERAGA, H. A., J. Biol. Chem., 227, 1043 (1957).

KLINE. D. L.. J. Biol. Chem.. 204,949 (1953). GREEN, N. G., AND NEURA~H, I?., J.. Bioi. Chem., 204, 379

(1953). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem., 212, 507

(1955). KUNITZ, M., J. Gen. Physiol., 30,291 (1947). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

R. J., J. Biol. Chem., 193, 265 (1951). VAN SLYKE, D. D., AND CULLEN, G. E., J. Biol. Chem., 19,

141, 211 (1914). CUNNINGHAM, L. W., JR., J. Biol. Chem., 207,443 (1954). GREEN, N. H., AND NEURATH, H., in H. NEURATH AND K. C.

BAILEY (Editors), The proteins, Vol. IIB., Academic Press, Inc., New York, 1954, p. 1067.

29.

30. 31.

32.

33. SIUITH, E. I;., I&MEL, J. R., AND %ROW~, b. M., J: Biol. Chem., 207, 533 (1954).

34. 35.

STOCKELL, A., AND S~~ITH, E. L., J. Biol. Chem., 227, 1 (1957). NEURATH, H., Ann. N. Y. Acad. Sci., 68, 11 (1957).

36. GREEN, N. M., J. Biol. Chem., 206, 535 (1953). 37. TROLL, W., SHERRY. S.. AND WACHMAN. J.. J. Biol. Chem..

208, 85 (i954). ’ I ,

38. GLADNER, J. A., AND LAKI, K., Arch. Biochem. Biophys., 62, 501 (1956).

39.

40. 41. 42.

43. 44.

45.

46.

47.

48.

49. 50.

51.

52. 53. 54.

55.

56.

KI~IMEL, J. R., AND SMITH, E. L., J. Biol. Chem., 207, 515 (1954).

SHERRY, S., AND TROLL, W., J. Biol. Chem., 208, 95 (1954). HALDANE, J. B. S., AND STERN, K., Allgemeine Chemie der

Enzyme, Theodore Steinkopf, Dresden and Leipzig, 1932, p. 119.

CUNNINGHAM, L. W., JR., TIETZE, F., GREEN, N. M., AND NEURATH, H., Discussions Faradau Sot.. 13. 58 (19531.

MILLER, K: D., AND VAN VUNAKIS, H., J. Biol. Chem., 223, 227 (1956).

RASMUSSEN, P. S., Biochim. et Biophys. Acta, 16, 157 (1955). FINKLE, B. J., AND SMITH, E. L., J. Biol. Chem., 230,669 (1958). ALKJAERSIG, N., FLETCHER, A. P., AND SHERRY, S., J. Biol.

Chem., 233, 86 (1958). ASTRUP, T., Blood, 11, 781 (1956). ALKJAERSIG, N., FLETCHER, A. P., AND SHERRY, S., J. Biol.

Chem., 233, 81 (1958). MOUNTER, L. A., AND SHIPLEY, B. A., J. Biol. Chem., 231,

855 (1958). GLAD~E~,~: A., AND NEURATH, H., Biochim. et Biophys. Acta,

9, 335 (1952). VALLEE, B. L., AND NEURATH, H., J. Biol. Chem., 217, 253

(1955). SINGER, T. P., AND HOFSTEE, B. H. J., Arch. Biochem., 18,

229 (1948). SINGER, T. P., J. Biol. Chem., 174, 11 (1948). NACHMANSOHN, D., AND WILSON, I. B., in S. P. COLOWICK

AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. I, Academic Press, Inc., New York, 1955, p. 647.

SMITH, E. L., AND PARKER, M. J., J. Biol. Chem., 233, 1387 (1958).

SMITH, E. L., J. Biol. Chem., 233, 1392 (1958). GUTFREUND, H., Discussions Faraday Sot., 20, 167 (1955). SCHWERT, G. W., AND EISENBERQ, M. A., J. Biol. Chem., 179,

665 (1949). MCDONALD, C. E., AND BALLS, A. K., J. Biol. Chem., 221,993

(1956). AUIXJSTINSSON, K. B., in J. B. SUMNER AND K. MYRBXCK

(Editors), The enzymes, Vol. I, Part 1, Academic Press, Inc., New York, 1950, p. 443.

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from

technical assistance of Albert R. FrazierCharles J. Martin, Julius Golubow, A. E. Axelrod, Albert R. Frazier and With the

Enzymes-Nitrophenyl Ester by VariouspThe Hydrolysis of Carbobenzoxy-l-tyrosine

1959, 234:1718-1725.J. Biol. Chem. 

  http://www.jbc.org/content/234/7/1718.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/234/7/1718.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on January 14, 2020http://w

ww

.jbc.org/D

ownloaded from